TY - JOUR
T1 - Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereus β-lactamase II
T2 - A perturbed angular correlation of γ-rays spectroscopic study
AU - Paul-Soto, Raquel
AU - Zeppezauer, Michael
AU - Adolph, Hans Werner
AU - Galleni, Moreno
AU - Frere, Jean Marie
AU - Carfi, Andrea
AU - Dideberg, Otto
AU - Wouters, Johan
AU - Hemmingsen, Lars
AU - Bauer, Rogert
PY - 1999/12/14
Y1 - 1999/12/14
N2 - Cd-substituted forms of the Bacillus cereus metallo-β-lactamases (BCII) were studied by perturbed angular correlation of γ-rays (PAC) spectroscopy. At very low [Cd]:[apo-β-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-β-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-β-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn2E, ZnE, Cd2E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.
AB - Cd-substituted forms of the Bacillus cereus metallo-β-lactamases (BCII) were studied by perturbed angular correlation of γ-rays (PAC) spectroscopy. At very low [Cd]:[apo-β-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-β-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-β-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn2E, ZnE, Cd2E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.
UR - http://www.scopus.com/inward/record.url?scp=0033554858&partnerID=8YFLogxK
U2 - 10.1021/bi9911381
DO - 10.1021/bi9911381
M3 - Article
C2 - 10600111
AN - SCOPUS:0033554858
SN - 0006-2960
VL - 38
SP - 16500
EP - 16506
JO - Biochemistry
JF - Biochemistry
IS - 50
ER -